B cells should preferentially present to helper T cells only peptides derived from proteins to which they can secrete antibodies; it is therefore likely that membrane immunoglobulin (mIg) specifically delivers bound antigen to appropriate, and potentially unique, intracellular compartment(s) for degradation and class II MHC association. The primary objective of this project is to examine the mechanisms that regulate efficient intracellular targeting and processing of extracellular antigen, particularly that bound to mIg. 1. Membrane immunoglobulin-associated molecules and mIg motifs directing the intracellular targeting of B cell antigen receptors. Various antigen- specific human mIgM's transfected into the A2O (H-2d) murine B cell line have been previously characterized, including interaction with known mIg- associated molecules. Mutations of a highly conserved transmembrane tyrosine (tyr587) leads to mIg's which rapidly intend upon antigen ligation, but do not access the antigen processing compartment in murine A20 cells, even if the mIg's interact with Ig-alpha and Ig-beta sheath molecules. It is hypothesized that appropriate intracellular trafficking of mIg is directed by distinct molecules which associate with mIg after antigen binding or internalization; these associations may be directed by unique patterns of intracellular protein phosphorylation. Therefore, proteins and phosphoproteins uniquely associated with wild-type mIg after internalization (vs. mutated and/or non-internalized mIg) will be characterized. In addition, the role of specific transmembrane residues and motifs for appropriate intracellular targeting will be examined by site-directed mutation and transfection of murine H-2Kb into A2O cell lines. Ability of these constructs to rapidly internalize after anti-H2Kb ligation will be assessed by FACS analysis; ability of internalized H-2Kb- antibody complexes to access intracellular processing compartment(s) will be assayed by subsequent efficient presentation of internalized anti-H- 2Kb-ovalbumin (OVA) complexes to an OVA-specific T cell hybridoma. 2. Characterization of alternative intracellular processing sites in B cells. While a low-density compartment plays a central role in processing certain mIg-internalized antigens in A20 cells, other antigens and other B cell lines appear to require distinct high-density organelles. Therefore, wild-type mIg transfected into the murine TA3 cell line (I- A(k/d)) will be used to characterize intracellular pathways of processing and MHC II association for two model antigens, OVA and hen egg lysozyme (HEL); these proteins appear to have different processing requirements for the generation of MHC II-peptide complexes. Analysis will include novel assays of antigen processing and presentation using subcellular fractions, allowing identification of intracellular sites of antigen degradation vs sites of MHC II association. 3. Characterization of the processing compartment(s). Using subcellular fractions obtained from Percoll gradients, by free flow electrophoresis, by a novel micro-particulate ferro-fluid coupled to antigen, or by affinity chromatography using cytoplasmic tail tags, the enzymatic, biochemical, and antigenic characteristics of the processing compartment(s) will be determined.